(1) Field of the Invention
This invention is related to prostaglandin analogs and method of preparation thereof, as precursors for the synthesis of oligomeric mixtures showing biological activity with regard to restoration of oxidative phosphorylation in the degenerated mitocondria and more particularly to oligomerization of the Ethyl Analog having low molecular weights which give rise to oligomeric mixture having various components thereof biologically active in vitro.
(2) Description of the Prior Art
A new class of polymeric derivatives designated PGB.sub.x and the syntheses thereof are disclosed in U.S. Pat. No. 4,153,802 issued May 8, 1979 to David Polis et al, which have the unique property of restoring the in vitro oxidative phosphorylation ability of isolated degraded mitochondria. Furthermore, synthesis of prostaglandin analogs including the Ethyl Analog as defined below and related compounds is disclosed in U.S. Pat. No. 4,338,466 issued July 6, 1982 to George L. Nelson. All the above-identified patents are incorporated herein by reference. The analogs used in the syntheses are prostaglandins such as PGB.sub.1, 13-14-dehydro-PGB.sub.1 and 15-keto-PGB.sub.1 methyl ester, each having a relatively complex molecular structure resulting in oligomeric derivatives which are not amenable to structural elucidation by conventional spectroscopic techniques necessary for defining the structure-activity relationships. Attempts by a number of research groups to resolve this complex mixture of oligomeric derivatives into individual components retaining biological activity have been unsuccessful.
Conversion of prostaglandin analogs such as 3-(trans-3-keto-1-pentenyl)-2-ethyl-2-cyclopentenone; hereinafter referred to as Ethyl Analog or E.A. into a higher molecular weight distribution bicarbonate soluble oligomeric mixture by treatment with ethanolic potassium hydroxide (KOH solution diluted with ethanol) with exposure to atmospheric oxygen has been tried by our group. These prostaglandin analogs are represented by: ##STR1## where R.sub.1, and R.sub.2 are members of the alkyl group. When R.sub.1 and R.sub.2 are CH.sub.2.CH.sub.3 each, the analog is called the Ethyl Analog (E.A.). However, the Ethyl Analog was oligomerized by treatment with ethanolic potassium hydroxide over a seven day period to give complex crude oligomeric mixture that was ca. 60 percent soluble. Both the bicarbonate soluble and insoluble fractions obtained from the crude oligomeric mixture were fractionated on Sephadex LH-20, a substrate for size exclusion chromatography. Mitochondrial activity in the protection of oxidative phosphorylation was observed for both the bicarbonate soluble and insoluble fractions with generally higher activity being observed in the bicarbonate soluble fractions. Most notably, inhibition of mitochondrial activity at higher concentrations, as is observed in the case of PGB.sub.x derived from 15-keto-PGB.sub.1, was not observed for the oligomeric mixture derived from the Ethyl Analog. The activities observed for the oligomeric mixture derived from the E.A. were generally lower than those observed for 15-keto-PGB.sub.1 derived PGB.sub.x at concentrations where PGB.sub.x exhibited maximum mitochondrial activity. However, at higher concentrations a greater protection was afforded by oligomeric fractions derived from the Ethyl Analog than that in the case of PGB.sub.x. The mitochondrial activity for both the bicarbonate soluble and insoluble fractions derived from Sephadex LH-20 (substrate for size exclusion chromatography) chromatography was distributed throughout the fractions.
Although the above-indicated results held much potential, several serious problems remained in this method for conversion of the Ethyl Analog (E.A.) into a bicarbonate soluble oligomer possessing the ability to protect isolated mitochondria against the loss of oxidative phosphrylation. The problems were generally associated with the severe conditions (7-day treatment with ethanolic KOH) used for the conversion to a bicarbonate soluble oligomeric mixture that was a very complex mixture and not readily amenable to structural elucidation by spectroscopic methods. For this reason, a series of experiments needed to be carried out to find out how the bicarbonate insoluble oligomer fraction can be maximized while reducing the severity of the reaction conditions, what is the functionality which gives rise to bicarbonate solubility and what molecular changes are involved in oligomer formation. It was on the basis of this investigation that conditions for the conversion of the Ethyl Analog to a bicarbonate insoluble (neutral) oligomeric mixture, as described in subject patent application and for the formation of a bicarbonate soluble (acidic) oligomeric mixture, as described in my copending patent application, Ser. No. 492,087, filed May 6, 1984, were developed.
In summary, several aspects critical to the oligomerization of Ethyl Analog (E.A.) resulted from these investigations. It has been determined that sufficient exposure to oxygen is required for formation of the functionality responsible for bicarbonate solubility. This functionality has been identified as the carboxylic acid although the detailed mechanistic mode of formation of this acidic functional group from the neutral Ethyl Analog is not completely defined. Taking advantage of this information, the Ethyl Analog could be oligomerized to high conversions of bicarbonate soluble oligomer (greater than 80 percent) under relatively mild conditions (3-6 hours at 50.degree. C.) if sufficient exposure to atmospheric oxygen was provided.